1. Field of Invention
The present invention relates to a primer, and more particularly to a primer for detecting short-chained RNAs and its application thereof.
2. Description of Related Arts
Short-chain RNA plays important role in gene regulation in many biological, physiological and pathological processes. MicroRNA (miRNA) belongs to the class of RNA with the greatest number amongst short-chain RNA. Because the RNA of miRNA and its analogs DNA is very short, its discovery is relatively difficult and its existence is only discovered in recent years. In 1993, lin-4 is the first short-chain RNA with 22 nt being identified and is regulated through lin-4 messenger RNA (mRNA) of target gene, which has inhibition effect on starting of development and cell division in C. elegan in studies. Until 7 years later, more discoveries of short-chain RNAs with similar structure and functions in human cells are found, and it is determined that lin-4 is not an isolated member and is classified as a type of gene regulator, which is a member of microRNA (miRNA).
Afterwards, miRNA is discovered to have functional activities on different areas such as embryogenesis, development, angiogenesis, cell growth and apoptosis. It also plays a role in the course of a series of diseases such as cancer, immunodeficiency and viral infection. Lu et al. discovered that when compared to normal tissue, the miRNA expression in tumor cell always exhibits a low level. Then, miRNA expression profiles can determine low differentiation of tumor classification while the messenger RNA expression profiles from the same testing sample fails to provide an accurate result. Recently, Rosetta Genomics Ltd. have placed a series of miRNA cancer diagnostic kits into the market and claim that these diagnostic kits can accurately detect the origin of metastatic cancer cell.
Although the functions and activities of miRNA requires further research studies, more and more researches are focused on testing and application of these small molecules due to academic and medical diagnosis reasons. Recently, it is discovered that miRNA in body fluid is far more stable than its messenger RNA counterpart because of its short molecular chain and argonaute complex protection. There are many reports about the possibility of using serum or plasma miRNA expression profiles as diagnostic tools for cancer prognosis, cancer cell identification and classification.
Because the sequence is short, the nucleotide variation is small and the lack of internal control reference, the most common messenger RNA (mRNA) detection method is not suitable for the detection of miRNA with 22 nucleotides. For examples, even though the method of quantitative reverse transcriptase PCR (qRT-PCR) is the golden standard for testing in messenger RNA (mRNA) expression and expression profiles, identification methods for miRNA mostly employ short-chain RNA clone screening and expression profiles includes deep sequencing, liquid or solid phase hybridization or RT-PCR based microarrays. From the technical view point, miRNA RT-PCR has already become the most accurate and sensitive method in relation to miRNA expression profiles and identification. Accordingly, RT-PCR single-plex assay is used to verify the accuracy of the data from microarrays.
Reverse transcript refers to the cDNA synthesis reaction with RNA template under the catalysis of reverse transcriptase. Annealing and complementary of oligonucleotides to RNA chain as the starting point, then the 5′ terminal to 3′ terminal of cDNA, based on the complementary principles with template RNA bases, goes through the synthesis process sequentially. Through the base complementary process in the matching region, the primer and the RNA template can form a double-stranded complex, which is the necessary requirement for the start (trigger) of reverse transcript reaction. In general, in order to provide sufficient Tm and specificity so as to ensure a double-strand complex specifically formed between primer and target (not other RNA) RNA template in a tightly and stably manner, the primer usually has a length of 20-30 nt. Short-chain RNA, in particular miRNA, is a type of oligonucleotides which has a length of 18-30 nt in average. Because its length is too short, conventional primers cannot be used to trigger the formation of cDNA. In order to improve the Tm of short-chain oligonucleotide of this type, a few types of modifications can be employed. For examples, LNA oligonucleotides can be used to connect a longer and universal oligonucleotides connector to the target RNA, polyA polymerase is used to increase the length of miRNA, and primer with stem-loop structure can be used.
At present, a few and completely different types of primers and qRT-PCR kits for miRNA detection are existed in the market. The most highly recognized product is the miRNA expression profiling and detection kit from Applied Biosystems Corporation, that this product features the use of a unique stem-loop RT design. Dr Chen in Applied Biosystems established a stem-loop primer which includes an ABI unique stem-loop structure which improves short-chain RNA detection in which the base stacking effect of stem-loop primer increases the thermodynamic stability of the fully binding pairs of target RNA and probe, then increases the efficiency of RT reaction. The detection sensibility is greater than that of the linear primer by 100 times.
According to the preferred embodiment of the present invention, the “stem-loop primer” of the present invention refers to the primer with the above unique stem-loop structure. Although it is unclear how the stem-loop primers improve the specificity and efficiency of reverse transcription, its inventor claimed that primers with stem-loop structure can increase the value of hybridization temperature (Tm) of short probe through the nucleotide bases stacking effect, and can selectively promote reverse transcription of short-chain RNA. It is worth mentioning that according to the experiments of the present invention, when the template is a synthetic RNA with no internal initiation sites, the initiation effect of the linear primer is at least as well as that of a stem-loop primer. Also, no additional experiments support this theory that this stem-loop structure will increase the accuracy of RT reaction. Therefore, it is hard to understand how this nucleotide bases stacking effect can increase the efficiency of reverse transcription. It is possible that the stem-loop primer has inhibition effect on the hybridization of target sequence (internal trigger), which forms part of the reason that this kind of primer can increase the detection of short-chain RNA efficiency.
Ribonucleic acid (RNA) can serve as a template for reverse transcription to synthesize single-stranded cDNA by following Watson-Crick base-pairing rule. This type of reverse transcription requires a small fragment of oligonucleotide (the primer) to bind to the complementary binding point of the template to form a stable duplex serving as the starting point of synthesis. Once the duplex is formed, even very short-chain oligonucleotide with only a few number of nucleotide bases is sufficient for effective initiation of DNA polymerization. In a certain type of reaction system, the initiation efficiency of a RT reaction depends on the stability of the primer-RNA duplex while temperature has a great impact on the stability of the duplex. The melting temperature (Tm) of a primer-RNA duplex refers to the temperature at which half of the primer-RNA duplex is dissociated. The primer Tm depends on the primer length and the composition of the primer-RNA duplex. When binding to a complementary target, a fragment of oligonucleotide with a length of 20-30 nt is sufficient to provide the required thermostability for the primer-RNA duplex which is required for starting the RT reaction. A lower temperature is required for shorter oligonucleotide primer in order to fulfil the stability of the duplex. Under the condition of a lower temperature, the enzymatic activity is relatively lower and the efficiency of DNA polymerization reaction is very low, while the number of potential target sites is increased and the specificity of DNA polymerization reaction is very low. Meanwhile, when under the condition of a lower temperature, polynucleotide will fold to form a very strong secondary structure, causing the product of RT to become shorter when compared to the product under the condition of a higher temperature. The length of oligonucleotide does not have significant effect on RT initiation efficiency and almost all of the commercial products of RT kit select random hexamer as the primer for synthesis of the first DNA strand. When using this type of short oligonucleotide primer, internal initiation is the biggest problem while the secondary structure of short-chain polynucleotide template will not become the major problem.
The critical factors in effective primer design for multi-target short-chain RNA is to provide solutions to avoid internal initiation and formation of primer dimer while increasing the initiation accuracy at the terminal location. The oligonucleotide primer with a length of 20-30 nt can initiate the synthesis of specific fragments, while starting PCR or RT at 37° C. or above can provide sufficient complexity and thermostability. However, since the length of this type of oligonucleotide is too long, it is not suitable for use in short-chain RNA reverse transcription with microRNA (miRNA) serving as template. In view of the identification and analysis of miRNA, the difficulty lies on the big challenge on thermodynamics in relation to the length of the miRNA. A relatively short oligonucleotide can serve as a primer under a relatively low temperature condition and no adverse effect will be produced on the initiation efficiency. However, if the length is extremely short, then the loss of complexity cannot be avoided. The loss of a high level of this type of complexity will increase the number of recognition sites (internal initiation sites) within the template nucleotide and cause the problem of non-specific amplification. As the number of internal initiation sites increases, the resources for reaction is consumed competitively, causing the overall RT efficiency and level of specificity to decreased dramatically. In short, because of internal initiation, short-chain linear primer cannot be used. The complicated miRNA biogenesis relates to pri-microRNA, pre-microRNA and matured microRNA, which further complicates its detection. In addition to internal initiation, the ability of the primer to distinguish mismatches in the primer-template at the 3′ terminal is another important factor affecting the RT efficiency.
As mentioned above, each of the above conventional technologies has its own limitations and restricts its applications' development. For example, LNA modification increases the Tm of oligonucleotide but adversely affects the enzymatic activities. Linear primer which has low specificity and internal initiation effect in the template nucleotide (which is not initiated from template's 3′ terminal) is not qualified to be used as primer for short-chain RNA detection. Stem-loop primer, which shows a preference to binding at the 3′ terminal of target RNA due to the existence of the stem-loop structure, does not have any advantages over other linear primers at least in view of the initiation efficiency.
According to the release of miRNABase v18, we learn that over 1500 different miRNAs are existed in human alone. The miRNA expressing profiling becomes more and more complicated. If no substantive changes in actual application is introduced, the use of stem-loop structure based primer for multi-target detection will become more and more difficult to operate and implement. Expression profiling based on oligonucleotide hybridization trades off sensitivity and preciseness. The result from expression profiling has to be further verified by RT-PCR. RT-PCR provides a quantitative and sensitive method for a few number of miRNA, but fails to process the increasing number of miRNA expression profiles. Now, in view of the surge of miRNA researches, a new technology for an accurate and high throughput method for miRNA expression profiling and analysis is urgently needed.